Dependency on host vitamin B12 has shaped Mycobacterium tuberculosis Complex evolution

Human and animal tuberculosis is caused by the Mycobacterium tuberculosis Complex (MTBC), which has evolved a genomic decay of cobalamin (vitamin B12) biosynthetic genes. Accordingly, and in sharp contrast to environmental, opportunistic and ancestor mycobacteria; we demonstrate that M. tuberculosis (Mtb), M. africanum, and animal-adapted lineages, lack endogenous production of cobalamin, yet they retain the capacity for exogenous uptake. A B12 anemic model in immunocompromised and immunocompetent mice, demonstrates improved survival, and lower bacteria in organs, in B12 anemic animals infected with Mtb relative to non-anemic controls. Conversely, no differences were observed between mice groups infected with M. canettii, an ancestor mycobacterium which retains cobalamin biosynthesis. Interrogation of the B12 transcriptome in three MTBC strains defined L-methionine synthesis by metE and metH genes as a key phenotype. Expression of metE is repressed by a cobalamin riboswitch, while MetH requires the cobalamin cofactor. Thus, deletion of metE predominantly attenuates Mtb in anemic mice; although inactivation of metH exclusively causes attenuation in non-anemic controls. Here, we show how sub-physiological levels of B12 in the host antagonizes Mtb virulence, and describe a yet unknown mechanism of host-pathogen cross-talk with implications for B12 anemic populations.

methylmalonic acid to succinic acid resulted in increased concentrations of methylmalonic acid which provided a particularly favorable climate for the multiplication of M. tuberculosis.They also found that macrophages were impaired in their ability to kill bacteria, suggesting that host B12 deficiencies altered immune function.
Lines 371 -378: It is evident that in the mouse strains used in this study, a host B12 effect on disease progression is clear.However, I think it is also premature to extrapolate results from this study to all mice (and from there to all humans, see above also).Notably, Smith et al 2020 showed that disease association with mutations in PPE, mut and bacA were observed in some mice strains, but not in others.This should also be made clear.Are the mouse strains used in this work associated with the groups assessed by Smith et al.?
I think the study claim (line 447-449) that they have reconciled historical, clinical, therapeutic and experimental evidence is exaggerated and should be worded more carefully.The study has certainly contributed greatly to the understanding of the ability of M. tuberculosis to access B12 in these mouse models, which might inform on aspects of human disease.
Reviewer #3 (Remarks to the Author): Applying the anemic-mice model, the Authors demonstrated that the depletion of B12 in serum affects the ability of Mtb to develop tuberculosis.Moreover, the Authors evaluated Mtb metE and metH mutant phenotype in an anemic mice model.However interesting, data confirm the previously reported studies demonstrating the B12-dependent phenotype of metE and metH mutants (e.g.Warner et al., 2007) and the effect of B12 transport inactivation (BacA-mutant) on chronic mouse infection (Domenech et al., 2009).Therefore, as such, the presented results do not introduce much novelty in the field of the B12 role in Mtb pathogenesis.Also, in certain other parts of the Results, the data repeats the previously published studies.Results-Part 1 "Bacteria causing TB in humans …." Lines 136-147; 155-156: The novelty of this part is very limited since the ability to uptake and inability to produce B12 by M. tuberculosis was, directly and indirectly, demonstrated in several previous studies: e.g.Warner et al., 2007;Domenech et al., 2009;Gopinath et al., 2013;Minias et al., 2021 along with the demonstration that metE promoter response in Mtb to the exogenous B12 in KO-cobIJ but not KO-bacA mutant (Minias et al., 2021).Moreover, as the Authors stated (Lines 142-143), the production of B12 by various strains of non-tuberculous mycobacteria was also previously demonstrated (Minias et al., 2021).The use of some new strains in the analysis is somehow interesting, however, it does not introduce enough novelty considering the chapter title (Line 119).Results-Part 2-3 "M.tuberculosis exhibits …" "B12 supplementation …" It's the most interesting part of the manuscript because of the anemic mouse model used by the authors.However interesting, data only confirm the previous, in vitro made observation that B12 deficiency affects the growth of Mtb strains.It could have been assumed that the same deficiency of B12 in vivo affects the survival/infection of Mtb.Moreover, it was also reported that Mtb bacA-KO-mutant (unable to uptake B12) is attenuated in chronic mice infection (Domenech et al., 2009).So, there is not much new information provided by authors regarding the completed experiments.
Results-Part 4 "The core B12-dependent …" Lines 217; 228-235; 265-267: The whole B12dependent transcriptome of the M. tuberculosis H37Rv, as well as the high level of B12-dependent downregulation of the prpR, prpCD/metE, have been previously demonstrated and discussed in detail (Pawelczyk et al., 2021;GEO database (GSE175812).The Authors do not cite and discuss this paper in any part of the manuscript.Again, the introduction of two more strains in the RNA-Seq analysis does not introduce much novelty to previously published transcriptomic results according to the core B12 response.
Lines 242-254 (Results) describe/discuss previously published data and should be transferred into the Discussion section.
Line 240: Defining a group of B12-downregulated Mtb genes as a "B12 regulon" is not supported by any additional molecular study (e.g.demonstration of direct B12 binding to the specific promoter regions or interaction between B12 and PrpR regulator).
Results-Part 5 "A M. tuberculosis …" Lines 272-274; 309-312: The in vitro B12-dependent phenotype of Mtb ∆metE and ∆metH mutants have already been published (Warner et al., 2007) therefore, in Results the Authors should focus on the in vivo studies only.Lines 282-286: Lmethionine transport ability in Mtb and M. canettii is an interesting original observation.
Results-Part 5 "Suppressive mutations …."The suppressor mutations identified in B12 riboswitch were an interesting observation made by the authors, but also quite expected in the event of inactivation of the metH gene.
In conclusion, the authors performed a lot of well-conducted experiments, however, mostly only confirmed the previous knowledge about the mechanisms of metabolism regulation in the presence of B12.The results obtained should be published in a more specialized journal.
Warner DF, et al. J Bacteriol. 2007 May;189(9):3655-9.Domenech P, et al. J Bacteriol. 2009 Jan;191(2):477-85.Gopinath K, et al. Open Biol. 2013 Feb 13;3(2)  We acknowledge your time and consideration during the revision of our manuscript.After carefully reviewing, answering and addressing the reviewer's comments, we are confident that the revised version of the study has been substantially strengthened.
We have reproduced the B12-dependent in vivo attenuation in mice adding additional time points, and including new mice and M. tuberculosis strains.Further, we have characterized some missing points related to the B12-dependent genes in M. tuberculosis.Lastly, we have, revised the main text, not only to address the reviewer's suggestions, but also to remove some exaggerated sentences.

Yours cordially
Jesús Gonzalo-Asensio (on behalf of the co-authors of the manuscript)

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): This is a very interesting study on M. tuberculosis and its ability to scavenge B12 during host infections.This study shows for the first time that virulence of Mtb, an auxotroph for B12, correlates with B12 plasma levels in mice, but that virulence of M. canettii a mycobacterium that is proficient in B12 biosynthesis is not dependent on B12 availability in the host.The authors then identify methionine biosynthesis as a possible reason for virulence attenuation because Mtb encodes a B12-dependent (MetH) and -independent (MetE) methionine synthase.Infection experiments using MetH and MetE mutants in anemic vs. normal mice indicate B12 dependent attenuation.Overall, the data presented is intriguing but not rigorous enough to make firm claims.
We acknowledge the overall positive impression of the reviewer praising the novelty of the study.We have conducted additional in vitro and in vivo experiments to improve the rigor of the presented data.
The main criticisms of the paper are: 1.No complementation of knockout strains was conducted and included in the assays.This is problematic because of potential secondary mutations e.g. in PDIM biosynthesis or compensatory mutations like the ones found in the MetH strain are not uncommon.
The reviewer is right, indeed from our previous works, we are confident that compensatory/secondary mutations might arise.However, this time, we decided not to include complemented strains based on two main claims: -In other virulence studies, the use of different M. tuberculosis strains (i.e. a wild type and a mutant) is the main differential factor to be interrogated.Thus, the emergence of undesired mutation(s) in the mutant strain could be ruled out by introducing complemented strain in the experiment.However, in the present study, we interrogate the phenotype of the same M. tuberculosis strain, assayed in the same mice background, being the B12 status of the animals the only differential factor to be interrogated.Accordingly, the assayed M. tuberculosis strain acts as a self-control in each experiment.In this same context, we decided to confirm that the differing B12 status of the animal is responsible for differences in M. tuberculosis virulence by conducting the B12 supplementation experiment shown in Figure 2.This experiment demonstrated that B12-deficient mice supplemented with B12 showed equivalent phenotype to control mice with normal B12 levels.
-By using metE and metH mutants, we anticipated that both strains should show complementary virulence phenotypes.While the metE mutant is more attenuated under B12 deficiency, the metH mutant is more attenuated when B12 is present.Showing two complementary phenotypes add robustness to the study by demonstrating the main hypothesis with two independent M. tuberculosis strains.
Nevertheless, our data support the absence of additional mutations in the metH mutant, at least in the context of the differential virulence in the variant B12 scenarios: -On one hand, the behavior of the metH mutant under B12 deficiency (Figures 4f,  4g) is indistinguishable from the wild type strain.Differences between the wild type and the metH mutant are exclusively observed under physiological B12 levels (Figures 4d,4e) due to the inhibition of metE by its B12 riboswitch.-The above-mentioned assumption is reinforced by the fact that complementation of the metH mutant with a B12-independent metE gene show the same phenotypes than the wild type in either control, or B12 deficient, mice (Figure 5).
For the metE mutant, we can rule out the emergence of impactful mutations leading to virulence attenuation, such those in PDIM synthesis, based on these observations: - The reviewer is right.We sincerely apologize for omitting this relevant reference, and their implications in the present work.We have conducted new experiments to demonstrate L-methionine transport in M. tuberculosis when growing in liquid and solid media.
First, we aimed at reproducing those experiments reported in Berney et al, who used a ∆metA auxotroph mutant able to recover growth in liquid media supplemented with 50 g/mL L-Met.We also expanded the L-Met range to include 25, 50 and 100 g/mL L-Met concentrations.We observed that the ∆metE mutant supplemented with the different L-Met concentrations showed equivalent growth in liquid media to the ∆metE mutant supplemented with B12, which acts as growth control (New Supplementary Figure 13).Similarly, the ∆metH mutant supplemented with B12 and different L-Met concentrations grew at comparable levels to the ∆metH mutant without B12, which serves as control (Supplementary Figure 13).Altogether, these observations are in line with those reported in Berney et al.
However, when examining the CFU growth on solid media under the above-mentioned conditions, we observed sharp differences relative to growth in liquid media.The ∆metE mutant was unable to growth even at the highest L-Met concentration (Supplementary Figure 13).Complementarily, the ∆metH mutant supplemented with L-Met and B12 did not completely recover the growth of the mutant cultured without B12.The observed growth of the ∆metH mutant supplemented with L-Met and B12 is indistinguishable from its growth under the sole presence of B12 (Supplementary Figure 13), indicative that those colonies are putative mutants in the metE riboswitch, as we will demonstrate in the answer for another reviewer's comment.methionine (Berney, 2015).Based on this observation we also confirmed the ability of the metE mutant to recover planktonic growth upon supplementation with L-methionine (Figure S13).Overall, this dependency on exogenous L-methionine of the metE mutant when growing on solid, but not in liquid media, is intriguing.We can hypothesize that either L-methionine bioavailability is higher in liquid media, facilitating its assimilation by M. tuberculosis; or that M. tuberculosis expresses L-methionine transport mechanisms exclusively under planktonic growth." Lines 304-306: As previously demonstrated with the metE mutant, we confirmed that assimilation of exogenous L-methionine in the metH mutant occurs under planktonic growth, but not during growth on agar plates (Figure S13).

3.
For mouse infection experiments in C57BL6 mice only one time point is shown, hence it cannot be determined if the small differences in CFUs measured are indeed B12 dependent or potentially stem from differences in inoculum size.Data with several time points, including a 24h harvest should be shown to prove the point that indeed Mtb.
We agree with the reviewer.Indeed, as requested in the following reviewer's comment, we have repeated all the mouse experiments including a 24-hour post-infection enumeration of lung CFUs.See the accompanying figure below.Please note that, for M. canettii C59, we previously optimized the bacterial inoculum to match equivalent phenotypes showed by M. tuberculosis, which explains the 100-fold increase after 24 hours post-infection with M. canettii C59, with respect to M. tuberculosis strains.

For M. tuberculosis H37Rv and M. canettii C59 wild type strains, we have also included a 2-month post-infection point, to gain insight into the B12-dependent phenotypes in the chronic infection phase (see below new figure panel 1g).
In addition to the 24-hour harvest, we also provide below the enumeration of the different glycerol stocks used to infect mice in the second round of experiments.

h 4 weeks
The manuscript is generally well written; however, the intro and discussion have sections that sound more like a review article.The story could be written in a more concise way.
Also discussion of what their findings mean for potential therapeutic strategies is missing, but could add value to the story.
We agree with the reviewer.The following paragraphs have been eliminated to provide a more straightforward message to the reader: -A plausible explanation for maintaining the B12 biosynthesis pathway in enteric Yersinia and Salmonella is that it is required for utilization of ethanolamine, a metabolite released from enterocytes during inflammation.Thus, the presence of B12-dependent ethanolamine respiration would enable enteropathogenic Enterobacteriaceae to utilize nutrients in the anaerobic environment of the gut and to outcompete other microorganisms from the microbiota.In this latter context, B12 is also necessary for Salmonella utilization of Concerning the therapeutic approaches, we initially mentioned the possible correlation between metformin administration and TB treatment, because a common side effect of metformin is related to lowering B12 levels in treated patients.However, the reviewer is right in pointing out that bacteria-related therapeutic approaches as blockading B12 transport, or inhibition of methionine metabolism, are attractive strategies against TB bacteria.We have included the following paragraph: -Lines 448-450: From our results, it is also appealing to propose new drugable targets in TB bacteria, based on either a chemical blockade of B12 transport, or inhibition of the methionine metabolism in M. tuberculosis.

Reviewer #2 (Remarks to the Author):
This study set out to discover whether host B12 status could affect progression of disease in a murine model of tuberculosis through interplay with Mycobacterium tuberculosis.The significance of this work is that it probes the access of M. tuberculosis to a desirable cofactor in what is generally considered to be a nutritionally poor space, and this informs on the metabolic consequences of this interplay on pathogenesis.
The work was carried out thoroughly and with attention to detail.All the results were provided for analysis, along with schema for experimental design.The methodology was clear and sufficiently detailed.In summary, the authors conducted a survey of current mutations in B12 biosynthetic genes amongst Mtb complex bacteria, which indicated ongoing decay in this pathway and demonstrated that members of the Mtb complex do not synthesize B12 although closely related ancestral lineages as well as the more distantly related environmental bacteria do.They also demonstrated that B12 uptake mechanisms were active for both cyanocobalamin and adenosylcobalamin in H37Rv, M. africanum and M. bovis.
In both SCID mice and C56BL/6 mice, a B12 deficient diet led to lower serum levels of B12.SCID mice were used for survival studies and C56BL/6 mice were used to assess bacterial burden at 4 weeks.B12-depleted mice survived longer than B12-replete mice, and the bacterial burden in C56BL6 mice was significantly lower at 4 weeks.In contrast, disease progression was identical regardless of host B12 status with M. cannetti, which synthesizes its own B12.
This finding was explored at a finer level with two different diets lacking B12, with substantially the same outcomes.The authors conclude that bacterial growth and pathogenesis was affected by host B12 status.The key question is whether the bacterium can directly access host B12, or if the loss of B12 affects how the host perceives the bacterium.
For these purposes, metE and metH mutant strains were constructed and assessed in mice for progression of disease.A ΔmetE mutant was significantly attenuated in SCID and C57BL/6 mice fed with a conventional diet, and even more so upon infection of anemic mice, which implies firstly that the bacteria could still access B12 from anemic mice to support metH function and growth, and secondly that even from B12-replete mice, the B12 available was not enough to support metH function.Accordingly, the bacterial burden of the metH mutant in mice was also lower and mice infected with metH mutant survived longer, implicating transport of B12 and partial repression of the metE riboswitch in this impairment.This was supported by infection studies with a constitutive metE expressing strain which was not attenuated.

Comments
Firstly, in S3g, the series +b12 and +B12/+Lmet are supposed to represent different conditions but seem to be duplicates of the same plates.
We sincerely apologize for this error, which arose during assembling this figure panel, and we acknowledge the perceptiveness of the reviewer.We have replaced the B12+L-Met panel with the correct picture, and we have carefully reviewed other similar panels to avoid undesired mistakes.
Otherwise, all the experiments were carried out carefully, all controls were present, and this work represents a substantial finding of general interest.However, I think some of the statements in the introduction are misleading, the conclusions may be overstated, and the limitations of the study have not been made clear as set out below.
We acknowledge the encouraging words of the reviewer, who highlighted the interest, the proper experimental design, and the inclusion of all experimental controls, in our work.We have rewritten the main text to avoid misleading/overstatements, as well as to reflect the limitations of the study.We are at your entire disposal to include additional changes in case that some unspotted sentences have not been amended.
lines 64-83: As most animals obtain B12 from the diet, are intestinal pathogens not expected to be able to source B12? Ie Where in the gut is B12 taken up by the host, and where do the bacteria reside?
This question was answered in lines 57-59 of the original version of the manuscript: However, even though some colonic bacteria produce Cbl, mammals are not able to uptake Cbl produced at this location, since the small intestine is the sole site of absorption.
We propose the following rewritting of this sentence to make a clear distinction between the site where bacteria reside, and the site of B12 absorption: Lines 57-59: However, even though some bacteria of the microbiota residing in the large intestine produce Cbl, mammals are not able to uptake Cbl produced at this location, since the site of B12 absorption is located in the small intestine lines 84-94: The references cited do not show that Listeria is sensing host B12 to regulate its virulence.B12 regulation of Listeria virulence is not an example of host B12-pathogen cross talk as Listeria is fully capable of de novo B12 biosynthetic capability (Vásquez et al, 2022; https://biolres.biomedcentral.com/articles/10.1186/s40659-022-00376-4). The insignificance of host B12 levels to pathogenesis to bacteria that possess the ability to manufacture B12 de novo is also shown by experiments conducted in the paper with M. canettii.

After carefully reading the manuscript of Vasquez et al. we are not completely confident
that Listeria is able (or not) to produce endogenous B12.In that manuscript, the authors quantified expression of cbi genes involved in B12 synthesis.However, a proper expression of these genes does not exclude the presence of non-functional mutations in the coding regions, similarly to the situation in M. tuberculosis.
Nevertheless, since this is a controversial observation -and another reviewer have recommended to rewrite the introduction in a more concise way-we consider more appropriate to eliminate the following paragraph: A solid molecular evidence for hostpathogen signalling mediated by B12 is provided by the intracellular pathogen Listeria monocytogenes.In this bacterium, a B12 riboswitch regulates the expression of a noncoding regulatory RNA, which in turns controls the expression of enzymes involved in ethanolamine utilization, that also require B12 as a cofactor.In fact, defects in ethanolamine utilization or in its regulation by the noncoding RNA attenuated Listeria virulence in mice.Another example is the presence of a B12 riboswitch in L. monocytogenes which controls transcription of a noncoding RNA involved in the regulation of the antisense gene pocR.In the presence of B12, this regulatory mechanism allows the PocR transcription factor to activate the expression of genes which mediate propanediol catabolism and are involved in pathogenesis.Together, both mechanisms integrate a way to sense host B12 to regulate the virulence of this intracellular pathogen.
The word 'deleterious" is used to describe the B12 biosynthetic pathway (line 151).Do the authors mean decayed?
The reviewer is right, we have replaced this word Although it is clear from this study (and previusly shown by Warner et al 2008) that sufficient external B12 pressure can select for riboswitch mutations in MetH mutants, such riboswitch mutants weren't directly identified from bacteria recovered from mouse lungs, and instead were only recovered after a laboratory-mediated stress passage through B12-containing media.The authors should make this clearer.I don't think evolution of a riboswitch mutation under these conditions is surprising either, as this has been previously demonstrated, so perhaps "surprising" (line 324) should be changed to "as expected".
We completely agree with the reviewer.Indeed, since we repeated mice experiments in answering reviewer 1, we decided to select colonies of the metH mutant recovered from mouse lungs to interrogate mutations in the metE riboswitch occurring in vivo.We selected 22 colonies of the metH mutant grown in vitro under B12 pressure, and 30 colonies from mouse lungs fed with normal diet.Results demonstrated that 17/22 colonies grown in vitro contained mutations in the riboswitch.In contrast, only one colony recovered from mouse lungs contained mutations in this region.We thank the reviewer for suggesting this experiment which suggest that the B12 pressure to select metE riboswitch mutations is lower in vivo than in vitro.

See below a screenshot showing the annealing of the selected sequences from mouse lungs colonies, indicating the unique metE riboswitch mutation found in vivo:
We have rephrased the paragraph as follows: Lines 311-323: We also sequenced the metE riboswitch region from 22 M. tuberculosis metH colonies grown on solid media supplemented with B12, and 30 colonies from lungs of mice infected with this strain and fed with normal diet.As expected, we found that 17/22 of the colonies grown on solid media contained mutations in the riboswitch (Figures 4H and S14).Surprisingly, a single colony from the mouse lungs contained mutations in this region, which might indicate that physiological levels of B12 do not impose a selective pressure as high as that observed in vitro.Mapping of the mutations to the predicted structure of the riboswitch demonstrated that these polymorphisms were regularly distributed, and different polymorphisms affecting invariant residues of B12 riboswitches arose independently in independent colonies (Figure 4I).This result suggests that suppressor mutations in the B12 riboswitch could alleviate the B12 repression of the metE gene, and favour the appearance of M. tuberculosis metH escape mutants when B12 is present, preferentially during growth in vitro.
The CDC1551 strain is a successful clinical strain that is compromised for metH activity and yet retains a functional B12 riboswitch and B12 transport.The paragraph (lines 392 -399) is therefore misleading since these experiments are not an example of the key role of host B12 in the evolution of riboswitch mutations as implied.
We agree with the reviewer; this paragraph is confusing in it actual form.We propose a rephrasing based on: -Demonstration that our M. tuberculosis metH mutant (equivalent to the CDC1551 strain) alleviates the host B12 pressure by accumulating mutations in the riboswitch in vivo.
-Demonstration that low concentrations of B12 ( 0 We are at the whole disposal of the reviewer to rewrite/clarify these concepts if they remain confusing. Contrary to this argument are the studies that find vegetarians are at a higher risk of active TB disease than non-vegetarians (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC473919/;https://jcp.bmj.com/content/jclinpath/41/7/759.full.pdf)Chanarin et al., found the incidence of tuberculosis in vitamin B12-deficient vegetarians was 133 per 1000, compared to 48 per 1000 in patients on varied diets.They suggested that in anemic patients, the failure to convert methylmalonic acid to succinic acid resulted in increased concentrations of methylmalonic acid which provided a particularly favorable climate for the multiplication of M. tuberculosis.They also found that macrophages were impaired in their ability to kill bacteria, suggesting that host B12 deficiencies altered immune function.
While these studies link vegetarianism with a higher incidence of TB, neither of these reports have explored the B12 status of the patients.Accordingly, we propose to cite both manuscripts in the context that the link between veganism/vegetarianism and TB should be examined carefully.The reviewer comment is very acknowledged, since it is necessary to alert the reader that, in the absence of proper biochemical markers, it is premature to associate veganism/vegetarianism with infectious diseases.
We propose to include the following paragraph: Lines 404-408 It is important to remark that veganism/vegetarianism does not necessarily involve a serum B12 deficit.Even if some studies linked the vegetarianism with a higher incidence of TB (Chanarin, 1988, Strachan, 1995), these studies do not report the B12 status of the patients.Accordingly, we cannot exclude B12-independent factors linked to vegetal diets that modulate the TB status in humans.
Lines 371 -378: It is evident that in the mouse strains used in this study, a host B12 effect on disease progression is clear.However, I think it is also premature to extrapolate results from this study to all mice (and from there to all humans, see above also).Notably, Smith et al 2020 showed that disease association with mutations in PPE, mut and bacA were observed in some mice strains, but not in others.This should also be made clear.Are the mouse strains used in this work associated with the groups assessed by Smith et al.?
This question is difficult to answer with precision.The CC (collaborative cross) mouse strains used in Smith et al. are a panel of recombinant-inbred lines generated by randomizing the genetic diversity of existing mouse resources (A/J, C57BL/6J, 129S1Sv/ImJ, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ).These 8 CC F0 lines were intercrossed to make F1s.Accordingly, the F1s strains used in the study of Smith et al. are mosaic population of F0 strains, as can be viewed in: http://csbio.unc.edu/CCstatus/index.py?run=CCV http://csbio.unc.edu/CCstatus/CCGenomes/One of the purposes of the study of Smith et al. was recapitulating the heterogeneity of TB disease, because current animal models do not reflect the human diversity.For this objective, Smith et al used the F1s CC mosaic mouse strains infected with a library of M. tuberculosis mutants for associating bacterial genetic requirements with host genetics and immunity.Thus, even if we have used C57BL/6 (which is a F0 strain), the genetic content of the intercross of this strain with another one will result in a mosaic of both genomes.It is possible that F1s CC mouse strains carry different polymorphisms affecting their susceptibility/resistance to M. tuberculosis.In a context of TB susceptibility depending on the B12 status of the mice, it is also possible that the F1s CC strains carry polymorphisms in B12 assimilation, or B12 metabolic genes.
Overall, it is beautiful to observe that in the context of the mouse host diversity, M. tuberculosis mutations in B12-related genes are down-selected in some F1s mouse (CC061, CC024, CC018, CC003, CC005, CC032, CC015, CC004, CC009, CC039), but not in others.In the context of TB disease depending on the B12 serum levels from the host, this would probably resemble the situation in humans since there is a heterogeneity in B12 serum levels between humans/populations.However, elucidating whether the polymorphisms of the CC061, CC024, CC018, CC003, CC005, CC032, CC015, CC004, CC009, CC039 are related (or not) to some B12 metabolic genes is clearly out of our knowledge.
The reviewer is right in advising that it is premature to extrapolate results from this study to all mice.However, it is important to note that the two mouse strains analyzed (SCIDwhich derives from the BALB/c background-, and C7BL/6) are genetically independent.Additionally, we performed a preliminary experiment in a third mouse background, DBA/2, which is known for being more sensitive to M. tuberculosis infection, and it represents a different background from SCID and C57BL/6.We observed >2-fold reduction in bacterial load when DBA/2 were fed a B12-deficient diet relative to the controls and results almost reached statistical significance (p=0.0581).Overall, even if we agree with the reviewer in her/his assumption, we are also confident that our results have been reproduced in three independent mice genetic backgrounds.
We propose the following rephrase of the paragraph mentioned by the reviewer: Lines 359-369: In Mycobacterium, a recent study used a combination of genetically diverse mice and a battery of M. tuberculosis mutants, with the objective of identifying bacterial virulence requirements in the context of a heterogeneous host genetics and immunity.Among their findings, bacA, mutB, and PPE2 mutants were negatively selected when infecting some mice strains.Of this pathway, bacA is a M. tuberculosis B12 transporter, mutB encodes one of the constituents of the B12-dependent MutAB methylmalonyl-CoA-mutase, and PPE2 is regulated by a B12 riboswitch.Even if this experimental design does not resemble our current study, this independent finding complementarily supports the importance of B12 during infection in murine models of TB.Nevertheless, it might be argued that our results obtained with two genetically independent, SCID and C57BL/6, laboratory mouse models, could not be extrapolated to all mice strains.
I think the study claim (line 447-449) that they have reconciled historical, clinical, therapeutic and experimental evidence is exaggerated and should be worded more carefully.The study has certainly contributed greatly to the understanding of the ability of M. tuberculosis to access B12 in these mouse models, which might inform on aspects of human disease.
We agree with the reviewer.This final paragraph has been rewritten as follows: In conclusion, our study, reinterprets the natural evolution of M. tuberculosis from its ancestor mycobacteria, shaped by the dependency on a host vitamin.Our findings using the mouse infection model might inform on aspects of human disease under different B12 scenarios, and might contribute to molecularly understand previous historical, clinical, therapeutical and experimental evidences Reviewer #3 (Remarks to the Author): Applying the anemic-mice model, the Authors demonstrated that the depletion of B12 in serum affects the ability of Mtb to develop tuberculosis.Moreover, the Authors evaluated Mtb metE and metH mutant phenotype in an anemic mice model.However interesting, data confirm the previously reported studies demonstrating the B12-dependent phenotype of metE and metH mutants (e.g.Warner et al., 2007) and the effect of B12 transport inactivation (BacA-mutant) on chronic mouse infection (Domenech et al., 2009).Therefore, as such, the presented results do not introduce much novelty in the field of the B12 role in Mtb pathogenesis.
We acknowledge this critical point of the reviewer.In fact, the mentioned literature, as well as related studies, are cited and discussed elsewhere in the manuscript.Below, we provide arguments about the novelty and added values of our study, which in our opinion represents a big leap in the context of M. tuberculosis pathogenesis in vivo: -   found that the M. tuberculosis bacA mutant is impaired in CNCbl uptake after short bation times with the vitamin relative to the wt strain (Figure 35).Incubation with CNCbl nger times resulted in a detectable, but very reduced ability to transport exogenous CNCbl pared to the wt strain (Figure 35A).AdoCbl uptake, conversely, was not affected by upting the bacA gene in M. tuberculosis (Figure 35B).This may be due to the high centration of AdoCbl added to the medium, which could have forced the entry of the vitamin ther mechanisms, or to the existence of other, as-yet-unidentified B12 uptake systems that sport exogenous AdoCbl inside the bacteria.In addition, it should be noted that in none of cultures without supplementation with the vitamin, neither in wt strain nor in its derived ants, intracellular B12 levels were detected (Figure 35), thus confirming the non-producing ogenous B12 phenotype of M. tuberculosis.2), 7 days (D.7) and 2 months (2m).The non-producing B12 phenotype is maintained in the bacA deficient mutant (H37Rv bacA::Kan) but vitamin uptake is not conserved after supplementing the medium with the CNCbl isoform.(B) Total B12 levels detected in cultures supplemented (+Ado) or not (-Ado) with AdoCbl for 2 days, 7 days and 2 months.The non-producing B12 phenotype is maintained in the bacA mutant as well as the vitamin uptake after supplementing the medium with AdoCbl isoform.All data represented are the mean and SD from two biological replicates.e phenotype observed in terms of AdoCbl uptake was reproducible in a second biological ica of the experiment where, in addition, the behaviour of the double mutant Rv1314c cA was also assessed.Again, AdoCbl uptake was not affected in the bacA mutant after a k of incubation (Figure 36).The novelty of this part is very limited since the ability to uptake and inability to produce B12 by M. tuberculosis was, directly and indirectly, demonstrated in several previous studies: e.g.Warner et al., 2007;Domenech et al., 2009;Gopinath et al., 2013;Minias et al., 2021 along with the demonstration that metE promoter response in Mtb to the exogenous B12 in KO-cobIJ but not KO-bacA mutant (Minias et al., 2021).
Moreover, as the Authors stated (Lines 142-143), the production of B12 by various strains of non-tuberculous mycobacteria was also previously demonstrated (Minias et al., 2021).The use of some new strains in the analysis is somehow interesting, however, it does not introduce enough novelty considering the chapter title (Line 119).
We agree with the reviewer, and indeed these references are cited elsewhere.Maybe the more direct evidence of B12 production in non-tuberculosus mycobacteria, but not in M. tuberculosis is reported by Minias et al. 2021. Indeed, we  from mouse lungs to interrogate mutations in the metE riboswitch occurring in vivo.We selected 22 colonies of the metH mutant grown in vitro under B12 pressure, and 30 colonies from mouse lungs fed with normal diet.Results demonstrated that 17/22 colonies grown in vitro contained mutations in the riboswitch.In contrast, only one colony recovered from mouse lungs contained mutations in this region.We greatly acknowledge the reviewer for suggesting this experiment which indicate that the B12 pressure to select metE riboswitch mutations is lower in vivo than in vitro; and accordingly, suppressor mutations are preferably selected in vitro.
See below a screenshot showing the annealing of the selected sequences from mouse lungs colonies, indicating the only mutation selected in vivo: We have rephrased the paragraph as follows: Lines 311-323: We also sequenced the metE riboswitch region from 22 M. tuberculosis metH colonies grown on solid media supplemented with B12, and 30 colonies from lungs of mice infected with this strain and fed with normal diet.As expected, we found that 17/22 of the colonies grown on solid media contained mutations in the riboswitch (Figures 4H and S14).Surprisingly, a single colony from the mouse lungs contained mutations in this region, which might indicate that physiological levels of B12 do not impose a selective pressure as high as that observed in vitro.Mapping of the mutations to the predicted structure of the riboswitch demonstrated that these polymorphisms were regularly distributed, and different polymorphisms affecting invariant residues of B12 riboswitches arose independently in independent colonies (Figure 4I).This result suggests that suppressor mutations in the B12 riboswitch could alleviate the B12 repression of the metE gene, and favour the appearance of M. tuberculosis metH escape mutants when B12 is present, preferentially during growth in vitro.
In conclusion, the authors performed a lot of well-conducted experiments, however, mostly only confirmed the previous knowledge about the mechanisms of metabolism regulation in the presence of B12.The results obtained should be published in a more specialized journal.We acknowledge the perception of the reviewer about the amount of work reported in this study, and the overall quality of the experiments performed.We have provided arguments throughout the reviewer's comments highlighting the novelty and added value of our work.We try to summarize the main arguments below: -Previous findings, with the exception of Domenech et al. 2009 (discussed above), exclusively focused on in vitro phenotypes.It is well known between microbiologists that correlation between in vitro and in vivo outcomes do not always occur, and it is possible to find contrasting phenotypes, especially in virulence studies.As a related example, mutants in the B12-regulated genes prpR, prpC, or prpD showed clear in vitro, or even ex vivo, phenotypes (Munoz-Elias, 2006, Masiewicz, 2012, Griffin, 2012, Pawelczyk, 2021), but failed to reproduce these phenotypes in vivo (Munoz-Elias, 2006, Eoh, 2014, Griffin, 2012), as the mutants behaved indistinguishable from their parent strains.-We provide for the first time an overview of the B12-dependent phenotypes in the M. tuberculosis Complex, non-tuberculous mycobacteria, and the ancestor-like mycobacteria.Thus, we provide a novel evolutionary and integrative story about the role of B12 in shaphing the virulence of TB (and non TB) bacteria.
Zaragoza, 21th December 2023 Manuscript reference number: NCOMMS-23-08005-T Manuscript title: Dependency on the host vitamin B12 has shaped the Mycobacterium tuberculosis Complex evolution Dear Reviewers, g) Pool of Exp. 1 and Exp. 2 independent experiments (n=6 mice/experiment; 12 mice/group in total).New Figures4d and 4f 24h and 2 weeks post-infection New Figures3h and 3j

Figure 35 .
Figure 35.Production and uptake kinetics of two B12 isoforms in in vitro cultures of the M. tuberculosis bacA mutant and its wt H37Rv strain.(A) Total B12 levels detected in cultures supplemented (+CN) or not (-CN) with CNCbl for 2 days (D.2), 7 days (D.7) and 2 months (2m).The non-producing B12 phenotype is maintained in the bacA deficient mutant (H37Rv bacA::Kan) but vitamin uptake is not conserved after supplementing the medium with the CNCbl isoform.(B) Total B12 levels detected in cultures supplemented (+Ado) or not (-Ado) with AdoCbl for 2 days, 7 days and 2 months.The non-producing B12 phenotype is maintained in the bacA mutant as well as the vitamin uptake after supplementing the medium with AdoCbl isoform.All data represented are the mean and SD from two biological replicates.
The new version of the manuscript contains 16 new, or revised, figure panels and 3 new supplementary figures.A complete list of the new experiments performed is provided below: 11) Demonstration of the B12-dependent phenotype in vivo using a third, independent, mouse strain.DBA/2 mice fed with control and B12-deficient diets were infected with M. tuberculosis and lung CFUs were enumerated 4 weeks Please find enclosed the manuscript text with the revised changes highlighted in yellow, as well as a new version of the figures and supplementary material.
All mouse experiments seem to have been done only once.This is concerning, because some of the differences shown (e.g.CFUs in BL6 mice) are very small.
canettii C59 infection of C57BL/6 mice fed with control, or B12 deficient, diets to enumerate lung CFUs after 24h, 4 weeks, or 8 weeks post-infection e) M. tuberculosis metE mutant infection of SCID mice fed with control, or B12 deficient, diets to enumerate survival.f) M. tuberculosis metE mutant infection of C57BL/6 mice fed with control, or B12 deficient, diets to enumerate lung CFUs after 24h or 4 weeks post-infection.g) M. tuberculosis metH mutant infection of SCID mice fed with control, or B12 deficient, diets to enumerate survival.h) M. tuberculosis metH mutant infection of C57BL/6 mice fed with control, or B12 deficient, diets to enumerate lung CFUs after 24h or 4 weeks post-infection.i) M. tuberculosis metH mutant-complemented infection of SCID mice fed with control, or B12 deficient, diets to enumerate survival.j) M. tuberculosis metH mutant-complemented infection of C57BL/6 mice fed with control, or B12 deficient, diets to enumerate lung CFUs after 24h or 4 weeks postinfection.We acknowledge this reviewer recommendation, since repetition of animal infections has resulted in reproducible results with higher statistical significances.The text has been modified to reflect results of the new animal experiments.A summary of these results is provided below, and additional information about each independent experiment (Exp. 1 and Exp. 2) can be accessed thought the Source data.xlsxfile: Groups a) and b) Pool of Exp. 1 and Exp. 2 independent experiments (n=6 mice/experiment; 12 mice/group in total).New Figure 1f.Groups c) and d) Group e) Pool of Exp. 1 and Exp. 2 independent experiments (n=6 mice/experiment; 12 mice/group in total).New Figures 3h and 3j , have counter evolved mechanisms to degrade itaconate and promote pathogenesis.Altogether, these evidences support the evolutionary arm-race of humans and pathogens to ensure access to B12. -At this point, we can theorize that adoption of agriculture 10.000 years ago might have alleviated the TB incidence by putatively decreasing B12 serum levels in Neolithic agrarian populations.It might be also possible that high burdens of TB in the past have selected human genetic variants associated with low B12 serum levels.Indeed, current genome wide association studies, has identified polymorphisms in European, Indian and Chinese populations associated with differential B12 serum concentrations, even if their associations with TB have not been yet established.-Even if reducing B12 serum concentrations under suboptimal levels in active TB patients is not an ethical possibility, maintaining B12 in a narrow physiological range could be an alternative strategy to accelerate recovery during the TB treatment.
1,2-propanediol, a catabolite produced by gut microbes fermenting fucose or rhamnose, common constituents of plant cell walls and intestinal epithelial cells lining the gut.-A solid molecular evidence for host-pathogen signalling mediated by B12 is provided by the intracellular pathogen Listeria monocytogenes.In this bacterium, a B12 riboswitch regulates the expression of a noncoding regulatory RNA, which in turns controls the expression of enzymes involved in ethanolamine utilization, that also require B12 as a cofactor.In fact, defects in ethanolamine utilization or in its regulation by the noncoding RNA attenuated Listeria virulence in mice.Another example is the presence of a B12 riboswitch in L. monocytogenes which controls transcription of a noncoding RNA involved in the regulation of the antisense gene pocR.In the presence of B12, this regulatory mechanism allows the PocR transcription factor to activate the expression of genes which mediate propanediol catabolism and are involved in pathogenesis.Together, both mechanisms integrate a way to sense host B12 to regulate the virulence of this intracellular pathogen.methylmalonyl-CoA-mutase, resulting in inactivation of the human-derived B12 pool.Both Itaconyl-CoA-mediated mechanisms, might well serve to reduce the B12 intracellular availability in infected macrophages as a strategy to fight intracellular infections.Conversely, intracellular pathogens as Y. pestis, or M. tuberculosis ) which again, represent an added value to this story.-Wecomplementthestudy ofWarner et al. byproviding evidence that M. tuberculosis, but not environmental (M.smegmatis), or ancestor-like (M.canettii) mycobacteria are able to uptake methionine when growing on solid media.These experiments required the construction and characterization of 7 different mutants in different mycobacteria, which in our opinion add value to our study.Further, since our in vivo experiments with the metE and metH mutants resembles the situation of these M. tuberculosis mutants growing on agar plates, we can also speculate about the inability of M. tuberculosis to assimilate methionine in vivo, or the insufficient intracellular methionine concentration to support M. tuberculosis in the animals.Please find below the new experiments performed to demonstrate these arguments:

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Neither the studies of Domenech et al, or Gopinath et al. directly demonstrate the ability of BacA to transport B12 in M. tuberculosis.These studies use indirect evidences of bacterial growth under the presence of B12.Thus, we performed direct B12 measurements in the newly constructed bacA mutant exposed to extracellular B12 to confirm, or discard, B12 transport in this strain.This experiment was performed at 2, 7 and 60 days incubation with B12, and we used the wild type strain as B12 assimilation control.Surprisingly, we observed that the bacA mutant is able to assimilate B12 when growing in laboratory media in a phenotype indistinguishable from the wild type (see this new result below).Thus, it is tempting to speculate that other mechanisms for B12 transport are present in M. tuberculosis H37Rv, at least during planktonic growth.-Basedon the previous observation, we reinforce the novelty of our findings.Being aware that B12 transport by BacA is a controversial result (due to either different B12 detection methods, strains used, cobalamin isoforms, or concentrations tested between studies), our experiments using the B12 deficient mouse models allows to unequivocally demonstrate the implications of B12 serum levels for virulence in vivo.The unique differential factor between mice groups fed with, or without B12 is the level of serum B12, and consequently, this allows to directly evaluate the impact of this vitamin on the virulence of wild type M. tuberculosis.Again, testing this phenotype in animal models is mandatory since bacterial properties in vitro are indistinguishable when cultured with or without B12.Further, the inclusion of the B12 producer, M. canettii, in our mouse infection ULTS AND DISCUSSION -Chapter 1

In vitro and in vivo phenotypic characterization of M. tuberculosis B12 uptake mutants
experiments represents novel results and provide an evolutionary focus of the story, which is missing in previous studies.

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We establish for the first time in M. tuberculosis research a mouse model of B12 deficit, being a pioneer work to molecularly demonstrate the B12 implications for M. tuberculosis virulence in vivo.-In this study, starting in 2014, we have used a collection of 15 wild type mycobacterial strains and we have constructed 8 recombinant strains.These bacteria have been used in diverse in vitro and in vivo studies to robustly, and complementarily demonstrate the reported phenotypes.We consider that our study not only provide novelty with respect to previous works, but it serves to valorize, confirm, and complement those findings by providing in vivo and mechanistic studies.